Refixation of photorespiratory ammonia and the role of alanine in photorespiration: Studies with15N

Summary Labelling experiments with¹⁵N glutamate and¹⁵N alanine were conducted using slices from oat leaves to investigate photorespiratory nitrogen metabolism. It is concluded from the labelling kinetics of glutamine that the refixation of photorespiratory ammonia primarily occurs by glutamine synthetase in the chloroplast. The labelling kinetics of glutamine with¹⁵N glutamate indicate that the chloroplastic and cytoplasmic glutamate pools do not exchange easily in oat leaf cells. Alanine was shown to be an important amino donor for photorespiratory glycine formation. This result is discussed with regard to a possible role of alanine in photorespiration. A modification to the scheme of photorespiratory nitrogen metabolism is proposed.     

Role of asparagine in the photorespiratory nitrogen metabolism of pea leaves

In pea leaves, much of the metabolism of imported asparagine is by transamination. This activity was previously shown to be localized in the peroxisomes, suggesting a possible connection between asparagine and photorespiratory nitrogen metabolism. This was investigated by examination of the transfer of ¹⁵N from the amino group of asparagine, supplied via the transpiration stream, in fully expanded pea leaves. Label was transferred to aspartate, glutamate, alanine, glycine, serine, ammonia, and glutamine (amide group). Under low oxygen (1.8%), or in the presence of α-hydroxy-2-pyridine methanesulfonic acid (an inhibitor of glycolate oxidase, a step in the photorespiratory formation of glyoxylate), there was a substantial (60-80%) decrease in transfer of label to glycine, serine, ammonia, and glutamine. Addition of isonicotinyl hydrazide (an inhibitor of formation of serine from glycine) caused a 70% decrease in transfer of asparagine amino nitrogen to serine, ammonia, and glutamine, while a 4-fold increase in labeling of glycine was observed. The results demonstrate the involvement of asparagine in photorespiration, and show that photorespiratory nitrogen metabolism is not a closed cyclic process.     

Metabolism of some amino acids in relation to the photorespiratory nitrogen cycle of pea leaves

¹⁵N-labelled (amino group) asparagine (Asn), glutamate (Glu), alanine (Ala), aspartate (Asp) and serine (Ser) were used to study the metabolic role and the participation of each compound in the photorespiratory N cycle ofPisum sativum L. leaves. Asparagine was utilised as a nitrogen source by either deamidation or transamination, Glu was converted to Gln through NH₃ assimilation and was a major amino donor for transamination, and Ala was utilised by transamination to a range of amino acids. Transamination also provided a pathway for Asp utilisation, although Asp was also used as a substrate for Asn synthesis. In the photorespiratory synthesis of glycine (Gly), Ser, Ala, Glu and Asn acted as sources of amino-N, contributing, in the order given, 38, 28, 23, and 7% of the N for glycine synthesis; Asp provided less than 4% of the amino-N in glycine. Calculations based on the incorporation of¹⁵N into Gly indicated that about 60% (Ser), 20% (Ala), 12% (Glu) and 11% (Asn) of the N metabolised from each amino acid was utilised in the photorespiratory nitrogen cycle.Abbreviations Ala alamine - Asn asparagine - Asp aspartate - Glu glutamate - MOA methoxylamine - Ser serine     

Regeneration in alfalfa tissue culture: stimulation of somatic embryo production by amino acids and N-15 NMR determination of nitrogen utilization

The production of somatic embryos in alfalfa (Medicago sativa L., cv Regen S) is increased 5- to 10-fold by alanine and proline. However, utilization of nitrogen for synthesis of protein from alanine, proline, glutamate, and glycine is not qualitatively different, even though the latter two amino acids do not increase somatic embryo formation. These determinations were made by ¹⁵N labeling with detection by nuclear magnetic resonance. Overall metabolism of the nitrogen of proline, alanine, glutamate, and glycine is also similar in two regenerating and nonregenerating genotypes with similar germplasm, except that the levels of free amino acids are consistently higher in the nonregenerating line. In addition, when regeneration is suppressed in either of the two regenerating lines, the level of intracellular free amino acids increases. This increased level of metabolites is the only direct evidence provided by analysis of nitrogen metabolism of differences between the regenerating and nonregenerating states in alfalfa.     

15N and inhibitor studies on the photorespiratory nitrogen cycle in maize leaves

A combination of inhibitor and ¹⁵N studies were used to investigate the photorespiratory nitrogen cycle in maize, a C₄ plant. Inhibitors used included isonicotinyl hydrazide which blocks the conversion of glycine to serine, methionine sulfoximine an inhibitor of GS and azaserine an inhibitor of GOGAT. Results from levels of ammonia and amino acids and the distribution of ¹⁵N into NH₃, serine, glutamine and glutamate indicated that the photorespiratory N-cycle occurs in this C₄ plant, but the rate of flux through this pathway is low as compared with that in C₃ plants.Abbreviations Aza azasering - fw fresh weight - GOGAT glutamate synthase - GS glutamine synthetase - INH isonicotinyl hydrazide - MSO methionine sulfoximine     

Metabolism of [15N]Alanine in the Ectomycorrhizal Fungus Paxillus involutus

Chalot, M., Finlay, R. D., Ek, H., and Söderström, B. 1995. Metabolism of [¹⁵N]alanine in the ectomycorrhizal fungus Paxillus involutus. Experimental Mycology 19, 297-304. Alanine metabolism in the ectomycorrhizal fungus Paxillus involutus was investigated using [¹⁵N]alanine. Short-term exposure of mycelial discs to [¹⁵N]alanine showed that the greatest flow of ¹⁵N was to glutamate and to aspartate. Levels of enrichment were as high as 15-20% for glutamate and 13-18% for aspartate, whereas that of alanine reached 30%. Label was also detected in the amino-N of glutamine and in serine and glycine, although at lower levels. Preincubation of mycelia with aminooxyacetate, an inhibitor of transamination reactions. resulted in complete inhibition of the flow of the label to glutamate, aspartate, and amino-N of glutamine, whereas [¹⁵N]alanine rapidly accumulated. This evidence indicates the direct involvement of alanine aminotransferase for translocation of ¹⁵N from alanine to glutamate. Alanine may be a convenient reservoir of both nitrogen and carbon.     

Nitrogen Metabolism in Senescent Flag Leaves of Wheat (Triticum aestivum L.) in the Light

Nitrogen metabolism was examined in senescent flag leaves of 90- to 93-day-old wheat (Triticum aestivum L. cv Yecora 70) plants. CO₂ assimilation and the levels of protein, chlorophyll, and nitrogen in the leaves decreased with age. Glutamine synthetase activity decreased to one-eighth of the level in young flag leaves. Detached leaves were incubated (with the cut base) in ¹⁵N-labeled NH₃, glutamate, or glycine in the light (1.8 millieinstein per square meter per second) at 25°C in an open gas exchange system under normal atmospheric conditions for up to 135 minutes. The ¹⁵N-enrichment of various amino acids derived from these ¹⁵N-substrates were examined. The amido-N of glutamine was the first ¹⁵N-labeled product in leaves incubated with ¹⁵NH₄Cl whereas serine, closely followed by the amido- and amino-N of glutamine, were the most highly ¹⁵N-labeled products during incubation with [¹⁵N]glycine. In contrast, aspartate and alanine were the first ¹⁵N-labeled products when [¹⁵N] glutamate was used. These results indicate that NH₃ was assimilated via glutamine synthetase and glutamate synthase activities and the photorespiratory nitrogen cycle remained functional in these senescent wheat flag leaves. In contrast, an involvement of glutamate dehydrogenase in the assimilation of ammonia could not be detected in these tissues.     

Nitrogen Assimilation in Mycorrhizas : Ammonium Assimilation in the N-Starved Ectomycorrhizal Fungus Cenococcum graniforme

Ammonium assimilation was followed in N-starved mycelia from the ectomycorrhizal Ascomycete Cenococcum graniforme. The evaluation of free amino acid pool levels after the addition of 5 millimolar NH₄⁺ indicated that the absorbed ammonium was assimilated rapidly. Post-feeding nitrogen content of amino acids was very different from the initial values. After 8 hours of NH₄⁺ feeding, glutamine accounted for the largest percentage of free amino acid nitrogen (43%). The addition of 5 millimolar methionine sulfoximine (MSX) to NH₄⁺-fed mycelia caused an inhibition of glutamine accumulation with a corresponding increase in glutamate and alanine levels.

Using ¹⁵N as a tracer, it was found that the greatest initial labeling was into glutamine and glutamate followed by aspartate, alanine, and ornithine. On inhibiting glutamine synthetase using MSX, ¹⁵N enrichment of glutamate, alanine, aspartate, and ornithine continued although labeling of glutamine was quite low. Moreover, the incorporation of ¹⁵N label in insoluble nitrogenous compounds was lower in the presence of MSX. From the composition of free amino acid pools, the ¹⁵N labeling pattern and effects of MSX, NH₄⁺ assimilation in C. graniforme mycelia appears to proceed via glutamate dehydrogenase pathway. This study also demonstrates that glutamine synthesis is an important reaction of ammonia utilization.

     

Evidence for the Glutamine Synthetase/Glutamate Synthase Pathway during the Photorespiratory Nitrogen Cycle in Spinach Leaves

Spinach leaf (Spinacia oleracea L.) discs infiltrated with [¹⁵N]glycine were incubated at 25°C in the light and in darkness for 0, 30, 60 and 90 minutes. The kinetics of ¹⁵N-incorporation into glutamine, glutamate, asparagine, aspartate, and serine from [¹⁵N]glycine was determined. At the beginning of the experiment, just after infiltration (0 min incubation) serine, and the amido-N of glutamine and asparagine were the only compounds significantly labeled in both light- and dark-treated leaf discs. Incorporation of ¹⁵N-label into the other amino acids was observed at longer incubation time. The per cent ¹⁵N-enrichment in all amino acids was found to increase with incubation. However, serine and the amido-N of glutamine remained the most highly labeled products in all treatments. The above pattern of ¹⁵N-labeling suggests that glutamine synthetase was involved in the initial refixation of ¹⁵NH₃ derived from [¹⁵N]glycine oxidation in spinach leaf discs.

The ¹⁵N-enrichment of the amino-N of glutamine was found to increase rapidly from 0 to 19% during incubation in the light. There was a comparatively smaller increase (4-9%) in the ¹⁵N-label of the amino-N of glutamine in tissue incubated in darkness. Furthermore the total flux of ¹⁵N-label into each of the amino acids examined was found to be greater in tissue incubated in the light than those in the dark. The above evidence indicates the involvement of the glutamine synthetase/glutamate synthase pathway in the recycling of photorespiratory NH₃ during glycine oxidation in spinach leaves.

     

Amino Acid Metabolism of Lemna minor L. : I. Responses to Methionine Sulfoximine

When Lemna minor L. is supplied with the potent inhibitor of glutamine synthetase, methionine sulfoximine, rapid changes in free amino acid levels occur. Glutamine, glutamate, asparagine, aspartate, alanine, and serine levels decline concomitantly with ammonia accumulation. However, not all free amino acid pools deplete in response to this inhibitor. Several free amino acids including proline, valine, leucine, isoleucine, threonine, lysine, phenylalanine, tyrosine, histidine, and methionine exhibit severalfold accumulations within 24 hours of methionine sulfoximine treatment. To investigate whether these latter amino acid accumulations result from de novo synthesis via a methionine sulfoximine insensitive pathway of ammonia assimilation (e.g. glutamate dehydrogenase) or from protein turnover, fronds of Lemna minor were prelabeled with [¹⁵N]H₄⁺ prior to supplying the inhibitor. Analyses of the ¹⁵N abundance of free amino acids suggest that protein turnover is the major source of these methionine sulfoximine induced amino acid accumulations. Thus, the pools of valine, leucine, isoleucine, proline, and threonine accumulated in response to the inhibitor in the presence of [¹⁵N]H₄⁺, are ¹⁴N enriched and are not apparently derived from ¹⁵N-labeled precursors. To account for the selective accumulation of amino acids, such as valine, leucine, isoleucine, proline, and threonine, it is necessary to envisage that these free amino acids are relatively poorly catabolized in vivo. The amino acids which deplete in response to methionine sulfoximine (i.e. glutamate, glutamine, alanine, aspartate, asparagine, and serine) are all presumably rapidly catabolized to ammonia, either in the photorespiratory pathway or by alternative routes.     

Interrelationships of Leucine and Glutamate Metabolism in Cultured Astrocytes

Abstract: The aim was to study the extent to which leu-cine furnishes α-NH₂ groups for glutamate synthesis via branched-chain amino acid aminotransferase. The transfer of N from leucine to glutamate was determined by incubating astrocytes in a medium containing [¹⁵N]leucine and 15 unlabeled amino acids; isotopic abundance was measured with gas chromatography-mass spectrometry. The ratio of labeling in both [¹⁵N]glutamate/[¹⁵N]leucine and [2-¹⁵N]glutamine/[¹⁵N]leucine suggested that at least one-fifth of all glutamate N had been derived from leucine nitrogen. At the same time, enrichment in [¹⁵N]leucine declined, reflecting dilution of the ¹⁶N label by the unlabeled amino acids that were in the medium. Isotopic abundance in [¹⁶N]-isoleucine increased very quickly, suggesting the rapidity of transamination between these amino acids. The appearance of ¹⁵N in valine was more gradual. Measurement of branched-chain amino acid transaminase showed that the reaction from leucine to glutamate was approximately six times more active than from glutamate to leucine (8.72 vs. 1.46 nmol/min/mg of protein). However, when the medium was supplemented with α-ketoisocaproate (1 mM), the ketoacid of leucine, the reaction readily ran in the “reverse” direction and intraastrocytic [glutamate] was reduced by ～50% in only 5 min. Extracellular concentrations of α-ketoisocaproate as low as 0.05 mM significantly lowered intracellular [glutamate]. The relative efficiency of branched-chain amino acid transamination was studied by incubating astrocytes with 15 unlabeled amino acids (0.1 mM each) and [¹⁵N]glutamate. After 45 min, the most highly labeled amino acid was [¹⁵N]alanine, which was closely followed by [¹⁵N]leucine and [¹⁵N]isoleucine. Relatively little ¹⁵N was detected in any other amino acids, except for [¹⁵N]serine. The transamination of leucine was ～17 times greater than the rate of [1-¹⁴C]leucine oxidation. These data indicate that leucine is a major source of glutamate nitrogen. Conversely, reamination of a-ketoisocaproate, the ketoacid of leucine, affords a mechanism for the temporary “buffering” of intracellular glutamate.     

Regulation by amino acids of photorespiratory ammonia and glycolate release from ankistrodesmus in the presence of methionine sulfoximine

Methionine sulfoximine induced release of ammonia from illuminated cells of Ankistrodesmus braunii (Naegeli) Brunnth, in normal air, but less in air enriched to 3% CO₂. In normal air, methionine sulfoximine also induced glycolate release. Addition of either glutamate, glycine, or serine suppressed glycolate release, whereas glutamate and glycine at the same time stimulated ammonia release. The results indicate that inhibition of glutamine synthetase and thereby inhibition of photorespiratory nitrogen cycling restricts the sink capacity for glycolate in the photorespiratory carbon cycle. An external supply of glutamate, glycine, or serine seems to stimulate glyoxylate transamination and thus partly restores the sink capacity. Calculations of total glycolate formation rates in air from glycolate and ammonia release rates in the presence of methionine sulfoximine and glutamate revealed values of approximately 20 micromoles glycolate per milligram chlorophyll per hour on the average. Similar calculations led to an estimated rate of photorespiratory ammonia release in air, in the absence of methionine sulfoximine, of about 10 micromoles per milligram chlorophyll per hour on the average, a value comparable to the primary nitrogen assimilation rate of 8 micromoles per milligram chlorophyll per hour.     

Two photosynthetic mechanisms mediating the low photorespiratory state in submersed aquatic angiosperms

The submersed angiosperms Myriophyllum spicatum L. and Hydrilla verticillata (L.f.) Royal exhibited different photosynthetic pulse-chase labeling patterns. In Hydrilla, over 50% of the ¹⁴C was initially in malate and aspartate, but the fate of the malate depended upon the photorespiratory state of the plant. In low photorespiration Hydrilla, malate label decreased rapidly during an unlabeled chase, whereas labeling of sucrose and starch increased. In contrast, for high photorespiration Hydrilla, malate labeling continued to increase during a 2-hour chase. Thus, malate formation occurs in both photorespiratory states, but reduced photorespiration results when this malate is utilized in the light. Unlike Hydrilla, in low photorespiration Myriophyllum, ¹⁴C incorporation was via the Calvin cycle, and less than 10% was in C₄ acids.

Ethoxyzolamide, a carbonic anhydrase inhibitor and a repressor of the low photorespiratory state, increased the label in glycolate, glycine, and serine of Myriophyllum. Isonicotinic acid hydrazide increased glycine labeling of low photorespiration Myriophyllum from 14 to 25%, and from 12 to 48% with high photorespiration plants. Similar trends were observed with Hydrilla. Increasing O₂ increased the per cent [¹⁴C]glycine and the O₂ inhibition of photosynthesis in Myriophyllum. In low photorespiration Myriophyllum, glycine labeling and O₂ inhibition of photosynthesis were independent of the CO₂ level, but in high photorespiration plants the O₂ inhibition was competitively decreased by CO₂. Thus, in low but not high photorespiration plants, glycine labeling and O₂ inhibition appeared to be uncoupled from the external [O₂]/[CO₂] ratio.

These data indicate that the low photorespiratory states of Hydrilla and Myriophyllum are mediated by different mechanisms, the former being C₄-like, while the latter resembles that of low CO₂-grown algae. Both may require carbonic anhydrase to enhance the use of inorganic carbon for reducing photorespiration.

     

Glucose and glycine metabolism in regenerating tobacco protoplasts: followed nondestructively by nuclear magnetic resonance spectroscopy

The metabolic states and the uptake and metabolism of [1-¹³C]glucose, [2-¹³C]glycine, and [¹⁵N]glycine in intact Nicotiana tabacum L. (cv Xanthi) mesophyll protoplasts were measured by ¹³C and ¹⁵N nuclear magnetic resonance spectroscopy. Changes in the concentration of metabolites during the first two days of culture in darkness were followed. Protoplasts isolated in 0.55 molar mannitol medium showed a drop in the concentration of all the intracellular metabolites during the first 28 hours of culture. Uptake of glucose and synthesis of glucose-derived metabolites were observed, indicating activity of glycolysis and the tricarboxylic acid cycle. Addition of glycine caused the accumulation of serine in dark cultured protoplasts, via the photorespiratory pathway. Glutamate dehydrogenase and glutamine synthetase activities in photorespiratory NH₄⁺ assimilation were observed. Glucose uptake and metabolism and cell division were inhibited by 3 millimolar glycine, suggesting that the accumulating serine or the release of ammonia during serine synthesis had toxic effects in this system.     

Photorespiratory nitrogen cycle – A critical evaluation

The photorespiratory nitrogen cycle proposed by Keys et al. (Nature 275: 741–743, 1978) involved formation of glycine by transamination of glyoxylate in the peroxisomes utilizing glutamate. Subsequently, glycine is oxidized to ammonia, serine and CO₂ in the mitochondria. The ammonia is reassimilated via the GS/GOGAT pathway generating glutamate. In this article, experimental evidence which suggests the occurrence of alternative mechanisms of glycolate and serine synthesis as well as of CO₂ and ammonia evolution is discussed. The problem of utilization of NADH coupled to ATP synthesis during photosynthesis is still unresolved, which complicates the glycine oxidation reaction in light. Further, factors are presented that determine the availability of amino donors in the peroxisomes and of amino acids viz., glycine, serine and glutamate for the operation of the photorespiratory N cycle. Recent evidence regarding the role of formate arising out of the reaction of glyoxylate with H₂O₂ in the regulation of photosynthetic electron flow in the Hill reaction, as well as of photorespiratory substrates functioning as carbon sources for the citric acid cycle in the light or for export to the growing tissues, suggests that the role of photo-respiration in plant metabolism needs to be reexamined.     

Ammonia Assimilation in the Roots of Nitrate- and Ammonia-Grown Hordeum Vulgare (cv Golden Promise)

¹⁵N kinetic labeling studies were performed on seedlings of Hordeum vulgare L. var. Golden Promise growing under steady state conditions. Patterns of label incorporation in the pools of nitrogen compounds of roots fed [¹⁵N]ammonium were compared with computer-simulated labeling curves. The data were found to be quantitatively consistent with a three-compartment model in which ammonium is assimilated solely into the amide-N of glutamine. Labeling data from roots fed [¹⁵N]nitrate were also found to be at least qualitatively consistent with the assimilation of ammonia into glutamine. Methionine sulfoximine almost completely blocked the incorporation of ¹⁵N label into the amino acid pools of barley roots fed [¹⁵N]nitrate. These observations suggest that ammonia assimilation occurs solely via the glutamine synthetase/glutamate synthase pathway in both nitrate- and ammonia-grown barley roots.     

Free amino Acid content and metabolic activities of setting and aborting soybean ovaries

Fruits of soybean (Glycine max [L.] Merr.) that are destined to abscise shortly after anthesis grow more slowly than fruits that will be retained. In this work, amino acid composition, protein metabolism, and nucleic acid metabolism were studied in setting and abscising soybean ovaries from anthesis to 6 days after anthesis to provide additional evidence of chemical processes associated with abscission. Principal free amino acids were asparagine, aspartic acid, glutamic acid, serine, and glutamine. Percent aspartate and glutamate declined as the ovaries grew, with aspartate declining more in abscising and glutamate more in setting ovaries. Percent glutamate was positively correlated to percent abscission throughout the period. Proline, serine, and leucine were positively correlated to abscission from 0 to 2 days after anthesis, whereas significant negative correlations were observed at these ages for ethanolamine and arginine. ⁷⁵Se fed as selenate and ¹⁴C fed as sucrose, glycine, and alanine were readily incorporated into soluble and insoluble proteins in a 24-hour in vitro incubation. Radioactivity of total proteins, expressed on a perovary basis, was negatively correlated with percent abscission and positively correlated with ovary weight. [¹⁴C]Glutamine and serine followed the opposite pattern, with greater protein labeling in abscising than in setting ovaries. When data were expressed as disintegrations per minute per milligram ovary fresh weight, protein labeling from alanine was seen to be significantly greater in abscising ovaries at anthesis and throughout the sampling period. Nucleic acid labeling from uridine was highly correlated to ovary weight; labeling from thymidine was greater in setting than abscising ovaries at anthesis and in abscising ovaries at later stages of development. It is concluded that abscising ovaries can continue amino acid metabolism almost up to the date of separation from the raceme, and that the involvement of alanine, glutamine, aspartate, glutamate, and other amino acids in soybean flower abortion deserves further study.     

Use of gaseous 13NH3 administered to intact leaves of Nicotiana tabacum to study changes in nitrogen utilization during defence induction

Nitrogen‐13 (t_1/2 9.97 m), a radioactive isotope of nitrogen, offers unique opportunities to explore plant nitrogen utilization over short time periods. Here we describe a method for administering ¹³N as gaseous ¹³NH₃ to intact leaves of Nicotiana tabacum L. (cv Samsun), and measuring the labelled amino acids using radio high‐performance liquid chromatography (HPLC) on tissue extract. We used this method to study the effects of defence induction on plant nitrogen utilization by applying treatments of methyl jasmonate (MeJA), a potent defence elicitor. MeJA caused a significant increase relative to controls in key [¹³N]amino acids, including serine, glycine and alanine by 4 h post‐treatment, yet had no effect on ¹³NH₃ incorporation, a process that is primarily under the control of the glutamine synthatase/glutamate synthase pathway (GS/GOGAT) in cellular photorespiration. We suggest that the reconfiguration of nitrogen metabolism may reflect induction of non‐photorespiratory sources of nitrogen to better serve the plant's defences.     

Studies on Amino Acid Metabolism in the Brain Using 15N-Labeled Precursors

The transfer of label from ¹⁵N-alanine and ¹⁵N-glutamate into amino acids in incubated brain slices has been followed using gas chromatography/mass spectrometry (GC/MS). ¹⁵N from alanine appeared in both amino and amide groups of glutamine more rapidly than into aspartate, glutamate and GABA, which were all labeled at similar rates. Maximum labelling of approx. 50% enrichment of these three metabolites was achieved in 3 hr. The ¹⁵N present in doubly-labeled glutamine exceeded that in the singly-labelled after 30 min. ¹⁵N from glutamate was rapidly transferred to aspartate and to alanine, with slower incorporation into glutamine and GABA. As was seen with labeling from alanine, doubly-labeled glutamine was higher than the singly-labeled species, also reaching some 50% enrichment in 3 hr. Depolarisation with 40 mM extracellular K⁺ caused a considerable reversal of the ratio of doubly- to singly-labeled glutamine species from both alanine and glutamate. The results are discussed in terms of the effects of depolarization on the glutamate/glutamine cycle.     

Effects of glycine hydroxamate, carbon dioxide, and oxygen on photorespiratory carbon and nitrogen metabolism in spinach mesophyll cells

The effects of added glycine hydroxamate on the photosynthetic incorporation of ¹⁴CO₂ into metabolites by isolated mesophyll cells of spinach (Spinacia oleracea L.) was investigated under conditions favorable to photorespiratory (PR) metabolism (0.04% CO₂ and 20% O₂) and under conditions leading to nonphotorespiratory (NPR) metabolism (0.2% CO₂ and 2.7% O₂). Glycine hydroxamate (GH) is a competitive inhibitor of the photorespiratory conversion of glycine to serine, CO₂ and NH₄⁺. During PR fixation, addition of the inhibitor increased glycine and decreased glutamine labeling. In contrast, labeling of glycine decreased under NPR conditions. This suggests that when the rate of glycolate synthesis is slow, the primary route of glycine synthesis is through serine rather than from glycolate. GH addition increased serine labeling under PR conditions but not under NPR conditions. This increase in serine labeling at a time when glycine to serine conversion is partially blocked by the inhibitor may be due to serine accumulation via the “reverse” flow of photorespiration from 3-P-glycerate to hydroxypyruvate when glycine levels are high. GH increased glyoxylate and decreased glycolate labeling. These observations are discussed with respect to possible glyoxylate feedback inhibition of photorespiration.